PROJECT SUMMARY Alpha protein kinase 3 (ALPK3, also known as MAK, MIDORI) is a novel atypical protein kinase highly expressed in cardiac muscle. Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy. Deficiency of ALPK3 in gene-trap mutant mice has been reported to cause cardiomyopathy. However, little is known as to the specific role of ALPK3 in cardiomyocytes, or molecular mechanisms by which loss of ALPK3 results in cardiomyopathy. Furthermore, it is yet to be determined whether ALPK3 is a true protein kinase or functions as a pseudokinase. To address the cardiac role of ALPK3, we have generated a floxed ALPK3 mouse line and used it to generate ALPK3 global knockout (gKO), as well as constitutive (cKO) and inducible (icKO) cardiac-specific knockout mouse models. Our preliminary data revealed that, similar to the published ALPK3 gene-trap mutant, ALPK3 gKO and cKO mice develop early onset cardiomyopathy. However, unlike the reported ALPK3 gene-trap mice, our ALPK3 gKO and cKO mice exhibited a more severe dilated cardiomyopathy (DCM) leading to premature lethality. We also observed that adult ALPK3 icKO mice develop DCM and heart failure. These observations strongly suggest that ALPK3 plays a critical role in both developing and adult cardiomyocytes. To test the kinase activity of ALPK3, we performed in vitro kinase assays using recombinant ALPK3 kinase domain. Surprisingly, we did not detect kinase activity. Moreover, we generated a novel ALPK3 knock-in mouse model in which the catalytic lysine (invariant lysine 1420) essential for phospho- transfer activity was mutated to arginine (ALPK3KR/KR), thereby disrupting putative ALPK3 kinase activity. ALPK3KR/KR mice did not display any cardiac abnormalities. Together, these observations indicate that the putative phospho-transfer activity of ALPK3 is not required for cardiac function. To study the role of the putative kinase domain, we generated a mutant mouse model in which the two Zn2+-coordinating cysteine residues critical for ?-kinase domain structure were mutated to alanine residues (ALPK3CA/CA). ALPK3CA/CA mice displayed DCM, albeit less severe and with delayed onset relative to the DCM observed in ALPK3 cKO mice. The cardiac phenotype of ALPK3CA/CA mutants indicates that a structurally intact putative kinase domain in ALPK3 is critical for cardiac function. Taken together, the foregoing observations lead us to the hypothesis that ALPK3 plays an essential role in regulating cardiac function, and that although devoid of catalytic activity, the putative kinase domain, and/or other domains of ALPK3, mediate protein interactions critical for cardiac function. Our Specific Aims are: 1. Elucidate the role of ALPK3 in developing and adult myocardium and 2. Decipher mechanisms underlying the requirement for the putative ALPK3 kinase domain, devoid of kinase activity, in cardiac function.